Interaction of vitronectin with collagen.

Purified human plasma vitronectin was demonstrated to bind to type I collagen immobilized on plastic as measured by enzyme-linked immunosorbent assay and by binding of 125I-radiolabeled vitronectin to a collagen-coated plastic surface. Vitronectin did not bind to immobilized laminin, fibronectin, or albumin in these assays. Vitronectin showed similar interaction with all types of collagen (I, II, III, IV, V, and VI) tested. Collagen unfolded by heat treatment bound vitronectin less efficiently than native collagen. Vitronectin-coated colloidal gold particles bound to type I collagen fibrils as shown by electron microscopy. Salt concentrations higher than physiological interfered with the binding of vitronectin to collagen, suggesting an ionic interaction between the two proteins. Binding studies conducted in the presence of plasma showed that purified vitronectin added to plasma bound to immobilized collagen, whereas the endogenous plasma vitronectin bound to collagen less well. Although fibronectin did not interfere with the binding of vitronectin to native collagen, vitronectin inhibited the binding of fibronectin to collagen. These results show that vitronectin has a collagen-binding site(s) which, unlike that of fibronectin, preferentially recognizes triple-helical collagen and that the binding between vitronectin and collagen has characteristics compatible with the occurrence of such an interaction in vivo.

Alternative surfaces for microcarrier culture of animal cells.

As part of an effort to broaden the applicability and efficiency of microcarrier cell culture various alternative new microcarriers were synthesized. The microcarriers were compared as substrates for the growth of several types of cells and with respect to binding of proteins from the culture medium. Cross-linked dextran has been found to be the most suitable material for a microcarrier matrix and was used as the matrix for the new microcarriers. One type of microcarrier was synthesized so that the charges necessary for cell attachment were present only in the surface layer of the microcarrier in the form of N,N,N-trimethyl-2- hydroxyaminopropyl groups. The resulting microcarriers had a very low capacity to bind proteins from the culture medium (e.g. albumin and IgG) and such proteins could be removed from the cultures more efficiently than when using previous microcarriers. A new principle was used for the development of the other type of microcarrier. A surface layer of cross-linked denatured collagen provided the surface for growth of cells. These microcarriers provided a "natural" substrate for cell growth and allowed improved attachment and spreading of cells with epithelial morphology. Harvesting cells from these microcarriers with proteolytic enzymes was more efficient than with previous microcarriers. The two alternative microcarriers should result in improved process efficiency and have a potential value in the preparation of live vaccines.

Harvesting and subculturing cells growing on denatured-collagen coated microcarriers (Cytodex 3).

Selecting correct procedures and conditions for harvesting cells was essential for successful subcultivation of cells when using the microcarrier culture technique. The proteolytic enzymes trypsin, Dispase and collagenase were compared with respect to the yield and the viability of Vero cells when harvested from Cytodex 3 microcarriers. Treatment with Dispase or trypsin resulted in similar viabilities but recovery after trypsin treatment was improved. The advantage of using collagenase was the improved plating efficiency of cells when subculturing to new microcarrier cultures. Pre-washing the confluent microcarriers with EDTA (0.02% w/v) improved the yield and the viability when harvesting with trypsin or collagenase. The stage of the culture cycle when cells were harvested did not influence relative recovery or viability. In contrast, significant differences in recovery and viability were found when harvesting cells grown in the presence of different batches of foetal calf serum. The results confirm that the choice of enzyme for harvesting depends upon the cell type and purpose of the culture.

The use of Cytodex 3 microcarriers and reduced-serum media for the production of nerve growth promoters from chicken heart cells.

Microcarrier cell culture provides an efficient method for the production of cell products. Cytodex 3 microcarriers were used for the production of an active nerve growth-promoting substance from chicken heart fibroblasts (1 degree -4 degrees cultures). Such cells release into culture medium a factor which stimulates the growth of nerve fibres from explanted ciliary, sympathetic and spinal neurons. Furthermore, culture in low-serum or serum-free media reduces the presence of contaminating proteins and facilitates the production and biochemical analysis of this factor. A mixture of DME/F 10 was supplemented with either 10% (v/v) foetal calf serum (FCS), 0.5% FCS, a low molecular weight fraction of FCS, (MW less than 10,000; prepared by dialysis) or different hormones and growth factors. Cells cultured in medium supplemented with insulin (I, 1 microgram/ml), transferrin (T, 25 micrograms/ml), human serum albumin (HSA, 2 mg/ml) and fibronectin (F, 10 micrograms/ml) (ITAF) in combination with 0.5% FCS or a low molecular weight fraction of FCS progressed through the cell cycle with normal kinetics and maximum DNA synthesis was after 20 h. The results were similar to those obtained with a supplement of 10% FCS alone. Media supplemented with insulin, transferrin, fibronectin and HSA in combination with dexamethasone (200 ng/ml) or epidermal growth factor (10 ng/ml) did not promote cell proliferation to the same extent. The fibroblasts proliferated on Cytodex 3 at a rate similar to cells grown on cell culture plastic and produced sufficient amounts of nerve growth-promoting substance for biological analysis. Production of this factor was generally associated with cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

The separation of harvested cells from microcarriers: a comparison of methods.

Two techniques are described which have been designed to separate harvested cells from microcarriers. The requirements of efficient recovery and high viability of the cells are met by both procedures. Differences both in size and density between cells and microcarriers allow separations which are based on differential centrifugation or filtration. After trypsinizing the cells from the microcarriers, separation was performed by either ; a) low-speed centrifugation on Ficoll-Paque or, b) filtration through nylon meshes. The methodologies for both techniques are presented. Up to 78% of the total cells were recovered by discontinuous gradient centrifugation using Ficoll-Paque. similar results were obtained when separating Vero, BS-C-1 and MRC-5 cells from Cytodex 1, Cytodex 2 and Cytodex 3 microcarriers. Equally high recoveries were obtained by filtration through an 88 micron pore size nylon mesh. Data are presented for the separation of both Vero and HeLa cells from all 3 types of microcarriers after filtration through 53 and/or 88 micron meshes. Greater than 92% viability of the recovered cells was consistently obtained and their growth properties upon subsequent reculturing were unaffected by either separation procedure. Differential sedimentation by unit gravity provides adequate cell recovery for many applications, but yields are significantly increased by using one or other of the methods described here. Both techniques are rapid and efficient. In addition, differential centrifugation provides a concentrated suspension of the recovered cells, while the nylon meshes for filtration are reusable and can be autoclaved at least 5 times.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of various laboratory scale culture configurations for microcarrier culture of animal cells.

When developing procedures for microcarrier culture it is important to use equipment which results in even suspension of microcarriers with minimal shear forces and which permits maximum growth of the culture. A variety of different laboratory scale culture systems were investigated including traditional magnetic spinner flasks, modified spinner flasks, cultures stirred with bulb-shaped rods and a fluid lift culture system. All systems were compared with respect to growth of either MRC-5 or Vero cells on Cytodex microcarriers. Although most of the earlier work with microcarriers has been performed with traditional magnetic spinner flasks, recently modified equipment resulted in better plating efficiencies and greater cell yields. Cultures stirred with a bulb-shaped rod and contained in flasks with contoured bases were particularly useful for cells having a low plating efficiency and provided thorough stirring of the medium with minimum shear forces. A fluid lift system based on upward perfusion of medium through a culture contained in a chromatographic column is described. This technique could provide a useful alternative for suspending microcarriers and provides for accurate in-line control of gas tensions.

Serum supplements and serum-free media: applicability for microcarrier culture of animal cells.

Several different combinations of serum supplements and also serum-free media were examined for their ability to support the growth of MRC-5 and Vero cells on Cytodex microcarriers. Greater economy of serum was achieved for routine microcarrier culture by selecting the type of serum supplement, on the basis of whether the supplement was to support attachment and growth of cells at low densities or growth of cells at later stages in the culture cycle. Further economy was achieved by altering the serum concentration according to the requirements of each stage of the culture cycle. More consistent results were obtained when batches of serum were selected on the basis of quality control tests with microcarrier culture as well as with monolayer culture. Low serum and serum-free media were able to support the growth of cells in microcarrier cultures. A mixture of DME/Medium 199 (50/50) containing foetal calf serum (0.5%, v/v), BSA and EGF supported growth of MRC-5 and Vero cells to nearly the same extent as DME/Medium 199 containing 10% (v/v) foetal calf serum. Serum-free media was used to achieve cell yields at least one half of those attained with serum-supplemented media. Maximum growth was obtained when DME/Medium 199 was supplemented with fibronectin, BSA, insulin, transferrin, putrescine, fetuin and EFG. Fetuin was omitted and replaced by dexamethasone and trace metals with only a slight reduction in cell yield. Fibronectin was essential in all serum-free media.

Alternative surfaces for microcarrier culture of animal cells.

As part of an effort to broaden the applicability and efficiency of microcarrier cell culture various alternative new microcarriers were synthesized. The microcarriers were compared as substrates for the growth of several types of cells and with respect to binding of proteins from the culture medium. Cross-linked dextran has been found to be the most suitable material for a microcarrier matrix and was used as the matrix for the new microcarriers. One type of microcarrier was synthesized so that the charges necessary for cell attachment were present only in the surface layer of the microcarrier in the form of N,N,N-trimethyl-2-hydroxyaminopropyl groups. The resulting microcarriers had a very low capacity to bind proteins from the culture medium (e.g. albumin and IgG) and such proteins could be removed from the cultures more efficiently than when using previous microcarriers. A new principle was used for the development of the other type of microcarrier. A surface layer of cross-linked denatured collagen provided the surface for growth of cells. These microcarriers provided a "natural" substrate for cell growth and allowed improved attachment and spreading of cells with epithelial morphology. Harvesting cells from these microcarriers with proteolytic enzymes was more efficient than with previous microcarriers. The two alternative microcarriers should result in improved process efficiency and have a potential value in the preparation of live vaccines.

Critical parameters in the microcarrier culture of animal cells.

The successful culture of over 60 different cell types on Cytodex TM1 microcarriers has enabled identification of parameters critical for obtaining high cell yields from microcarrier cultures. Careful control of the initial phase of microcarrier culture was found to be critical. Increasing cell density, reducing culture volume and reducing stirring rate during the initial phase all assisted in improving cell yields. Control of pH, inoculum condition, nutrient supply and serum quality, and elimination of mycoplasma were all vital for high cell yields. Knowledge of the in vitro growth properties of the cells made it easier to arrive at optimal conditions for microcarrier culture of each cell type.

Spectroscopic evidence for the formation of a 4-keto intermediate in the UDP-apiose/UDP-xylose synthase reaction.

Uridine diphospho-D-glucose (UDP-Glc) and UDP-methyl-D-glucuronate (UDP-GlcUAMe) have been shown to be competitive inhibitors for the UDP-apiose/udp-xylose synthase from cell suspension cultures of parsley. The apparent Ki values for these substrate analogues were of the same order of magnitude as the apparent Km value for the substrate UDP-D-glucuronic acid (UDP-GlcUA). The difference spectrum of the incubation mixture containing UDP-GlcUA, NAD+ and the highly purified enzyme showed a transient absorption with a maximum at 292 nm which disappeared upon addition of sodium hydroxide. The incubation with UDP-Glc or UDP-GlcUAMe also showed and NAD+-dependent absorption at 292 nm. However, in these cases a strong enhancement of the absorption at alkaline pH and a shift of the absorption maximum to longer wavelength were observed. Upon addition of 0-phenylenediamine to the enzyme incubation with UDP-Glc or UDP-GlcUAe a strong absorption with a maximum at respectively 335 and 315 nm appeared. The results show the transient formation of a 4-keto derivatives in the synthase reaction with the normal substrate UDP-GlcUA. The substrate analogues UDP-Glc and UDP-GlcUAMe can also be oxidized at C-4 by the enzyme in the presence of NAD+ to stable 4-keto derivatives which give rise to a strong absorption at alkaline pH or after reaction with o-phenylenediamine.